PCR

Question 1

What happens when DNA is heated?

Answer 1

It denatures

Question 2

What happens when DNA is renatured?

Answer 2

Complementary pieces of single-strand DNA can pair

Question 3

What are oligonucleotides?

Answer 3

Synthetic ssDNA that is 20 nucleotides long

Question 4

What is the purpose of oligonucleotides in PCR?

Answer 4

Oligonucleotides can bind to complementary ssDNA and serve as primers for in vitro DNA replication

Question 5

What is the purpose of PCR?

Answer 5

In vitro exponential amplification of a small region of DNA

Question 6

What are 2 limiting factors of PCR?

Answer 6

1. Must know the sequence at both ends of the region to be amplified
2. The region to be amplified can’t be bigger than 5 kb

Question 7

What are the materials required for PCR?

Answer 7

Template DNA
Taq DNA pol 
Left/forward oligo primers 
right/reverse oligo primers 
dNTPS 
Thermocycler machine

Question 8

What is thermocycling?

Answer 8

Denature DNA by heating to 95 degrees Celsius
Anneal DNA at 55 degrees Celsius, this includes oligo primers
Extend DNA at 72 degrees Celsius

Question 9

What is the result of thermocycling?

Answer 9

After 30 cycles the tube will contain:

• original template DNA
• some unused dNTPs and primers
• some variable-length DNA
• LOTS of constant length DNA

Question 10

What do we see when we run the results of PCR on gel?

Answer 10

Only the constant length DNA is visable

Question 11

What are some uses of PCR?

Answer 11

Amplify DNA for sequencing
Detect differences in the length of a gene
Determine a person’s genotype

Question 12

How do we determine a person’s genotype using PCR?

Answer 12

Isolate DNA

PCR

Question 13

What gel is used after PCR?

Answer 13

Agarose